SUBSTRATE SPECIFICITIES of TISSUE KALLIKREIN and T-KININOGENASE - THEIR POSSIBLE ROLE in KININOGEN PROCESSING

The present studies demonstrate the importance of subsite interactions in determining the cleavage specificities of kallikrein gene family proteinases. the effect of substrate amino acid residues in positions P3-P'3 on the catalytic efficiency of tissue kallikreins (rat, pig, and horse) and T-kininogenase was studied using peptidyl-pNA and intramolecularly quenched fluorogenic peptides as substrates. Kinetic analyses show the different effects of D-amino acid residues at P3, Pro at P'2, and Arg at either P'1 or P'3 on the hydrolysis of substrates by tissue kallikreins from rat and from horse or pig. T-Kininogenase was shown to differ from tissue kallikrein in its interactions at subsites S2, S'1, and S'2. As a result of these differences, Abz-FRSR-EDDnp with Arg at P'2 is a good substrate for tissue kallikreins from horse, pig, and rat but not for T-kininogenase. Abz-FRRP-EDDnp and Abz-FRAPR-EDDnp with Pro at P'2 (rat high molecular weight kininogen sequence) are susceptible to rat tissue kallikrein but not to tissue kallikreins from horse and pig. Arg at P'3 increased the susceptibility of the Arg-Ala bond to rat tissue kallikrein. These data explain the release of bradykinin by rat tissue kallikrein and of kallidin by tissue kallikreins from other animal species. Abz-FRLV-EDDnp and Abz-FRLVR-EDDnp (T-kininogen sequence) are good substrates for T-kininogenase but not for tissue kallikrein. Arg at the leaving group (at either P'1, P'2, or P'3) lowers the K(m) values of T-kininogenase while Val at P'2 increases its k(cat) values. the results indicate that the enzyme subsites S'1, S'2, and S'3 are important determinants for the substrate specificity of tissue kallikreins and T-kininogenase. the findings are also in agreement with the known species specificity of tissue kallikreins and the resistance of rat T-kininogen to tissue kallikreins.MED UNIV S CAROLINA,DEPT BIOCHEM & MOLEC BIOL,171 ASHLEY AVE,CHARLESTON,SC 29425ESCOLA PAULISTA MED SCH,DEPT BIOPHYS,BR-04023 São Paulo,BRAZILESCOLA PAULISTA MED SCH,DEPT BIOPHYS,BR-04023 São Paulo,BRAZILWeb of Scienc